A modified Western blot protocol for enhanced sensitivity in the detection of a membrane protein.
Membrane proteins, owing to their highly hydrophobic nature, have always posed a daunting challenge to biochemists and structural biologists working on the characterization of these "naughty" proteins. Here we describe a problem that we encountered in the immunodetection of a hemagglutinin (HA) epitope-tagged membrane protein, Hgt1p (high-affinity glutathione transporter from the yeast Saccharomyces cerevisiae), for which little or no signal was observed on the blots with monoclonal antibody, on following the standard Western blot protocol. The introduction of a single step that involved posttransfer incubation of the blots with sodium dodecyl sulfate (SDS)/beta-mercaptoethanol solution at 55 degrees C for 15 min enabled us to detect a strong, stable, and reproducible signal for the membrane protein.
Kaur
Jaspreet
J
Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036, India. jaspreet@imitech.res.in
Bachhawat
Anand K
AK
D015153:Blotting, Western / Q000379:methods*; D006388:Hemagglutinins / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012680:Sensitivity and Specificity
D006388:Hemagglutinins; D008565:Membrane Proteins